This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The regulation of sodium absorption by epithelial cells is key to understanding the pathological disruptions in many human diseases, yet it remains poorly understood. Sodium absorption via the epithelial sodium channel, ENaC, is believed to be regulated by the Cystic Fibrosis Transmembrane Conductance Regulator, CFTR. CFTR is a multifunctional protein that not only functions as a cAMP-gated chloride channel, but that also exists in a macromolecular signalling complex, interacting directly with scaffolding proteins, upstream modulators and downstream effectors (egs. the beta-adrenergic receptor and SLC26 transporter family members). On the other hand, direct molecular interactions between CFTR and ENaC have not been demonstrated. In general, the regulation of ENaC's net contribution to epithelial transport may be accomplished by changing its gating, its single channel conductance and/or the number of functional channels in the plasma membrane. The thyroid gland is an organ in which CFTR- transporter interactions at the level of the macromolecular signalling complex may influence ENaC activity critically. The disruption of those interactions may dysregulate ENaC and cause disease. The present proposal asks whether CFTR regulates epithelial sodium absorption by influencing a constitutive property of all cells, endocytosis, and hence changing the numbers of ENaC in the membrane. With regard to mechanism, + Does activation of CFTR normally cause cells to release ATP into the apical compartment? + Does ATP stimulate endocytosis? + How does endocytosis modulate net epithelial sodium absorption? To address this hypothesis, the proposed study will use primary porcine thyroid epithelial cultures, a robust, bidirectionally transporting epithelial preparation. The approach incorporates measurements of short-circuit current and employs specific pharmacologic tools in these measurements, as well as in fluorescence microscopic assays of endocytic uptake, cell surface biotinylation, and immunolocalization studies. The combination of several methodologies will enable clear answers to the posed questions to be drawn, leading to novel insights that in turn will clear a path toward future questions. It is envisaged that these studies will fill a gap in knowledge that presently impedes overall progress in understanding diseases as diverse as Pendred syndrome, cystic fibrosis and polycystic kidney disease.